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H1N1 swine influenza virus-resistant hemagglutinin protein monoclonal antibody, hybridoma cell line and antigen-capture ELISA kit

A hybridoma cell line and hemagglutinin protein technology, applied in the field of immunology, can solve the problems of high requirements for detection equipment and professional knowledge of personnel, potential biological safety hazards, and long time-consuming virus isolation

Inactive Publication Date: 2011-03-23
湖南出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation takes a long time, and at the same time there are biological safety hazards; RT-PCR and fluorescent RT-PCR methods have high requirements for detection equipment and professional knowledge of personnel, high cost, and are difficult to carry out in ordinary laboratories and grassroots. Therefore, the development of a direct detection The rapid, convenient, and field-use detection kit for H1N1 swine influenza virus in samples has strong practical value and broad application prospects, and is of great significance to the prevention and control of swine influenza

Method used

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  • H1N1 swine influenza virus-resistant hemagglutinin protein monoclonal antibody, hybridoma cell line and antigen-capture ELISA kit
  • H1N1 swine influenza virus-resistant hemagglutinin protein monoclonal antibody, hybridoma cell line and antigen-capture ELISA kit
  • H1N1 swine influenza virus-resistant hemagglutinin protein monoclonal antibody, hybridoma cell line and antigen-capture ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1, preparation of anti-H1N1 swine influenza virus specific monoclonal antibody

[0015] In the present invention, the monoclonal antibody is prepared using hybridoma technology, specifically as follows:

[0016] a. Animal immunity

[0017] The formaldehyde inactivated H1N1 subtype swine influenza virus was used to immunize Balb / c mice for three times.

[0018] b. Cell Fusion

[0019] Spleen cells from immunized mice were isolated and fused with SP2 / 0 myeloma cell line.

[0020] c. Screening of positive hybridoma cell lines

[0021] Add mouse peritoneal feeder cells to co-culture with fusion cells, and screen for positive strains.

[0022] d. Cloning Screening by Limiting Dilution

[0023] The positive wells were cloned by limiting dilution until a hybridoma cell line capable of stably secreting anti-H1N1 swine influenza virus hemagglutinin protein was obtained.

[0024] The invention obtains a hybridoma cell strain capable of stably secreting and highly...

Embodiment 2

[0027] Example 2, Development of H1N1 Swine Influenza Virus Hemagglutinin Protein Capture ELISA Detection Kit

[0028] 1. Composition of H1N1 Swine Influenza Virus Hemagglutinin Protein Capture ELISA Kit

[0029] The H1N1 swine influenza virus hemagglutinin protein capture ELISA detection kit of the present invention consists of: a solid phase carrier that has been coated with the monoclonal antibody of the present invention, that is, an ELISA plate, and horseradish peroxidase-labeled anti-H1N1 pig Influenza virus hemagglutinin protein polyclonal antibody, enzyme substrate reaction solution, positive and negative controls, washing solution and reaction stop solution.

[0030] 1) ELISA strip coated with anti-H1N1 swine influenza monoclonal antibody

[0031]Dilute the purified monoclonal antibody of the present invention to a concentration of 10 μg / mL with a coating buffer (0.05mol / L carbonate buffer, pH 9.6), and coat a 96-well EIA / RIA high-efficiency binding enzyme plate (Co...

Embodiment 3H1

[0062] Example 3 Specificity and Sensitivity Determination of H1N1 Swine Influenza Virus Hemagglutinin Protein Capture ELISA Detection Kit

[0063] 1) Specificity determination (the following experimental materials were inactivated at 56°C for 30 minutes)

[0064] In order to verify the specificity of the H1N1 swine influenza virus hemagglutinin protein capture ELISA detection kit of the present invention, according to Example 2, 2 strains of Newcastle disease virus samples (No. 1, 2), 2 strains of H3N2 virus samples (No. 3, 4) ), 1 strain of H5N1 virus sample (No. 5), 1 strain of H7N1 virus sample (No. 6) and 1 strain of H9N2 virus sample (No. 7) were tested. The results show that the detection OD value of the kit of the present invention for the above-mentioned protein samples is between 0.078-0.116 (Table 1), showing good specificity.

[0065] Table 1 Specific detection of the kit

[0066]

[0067] 2) Sensitivity determination

[0068] The H1N1 virus allantoic fluid w...

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Abstract

The invention discloses an H1N1 swine influenza virus-resistant hemagglutinin protein monoclonal antibody, a hybridoma cell line and an antigen-capture enzyme linked immunosorbent assay (ELISA) kit. The monoclonal antibody is secreted by a hybridoma cell of which the collection number is CCTCC C2010121, and has high titer and specificity. The developed antigen-capture ELISA kit comprises a solid-phase vector coated with the monoclonal antibody, the H1N1 swine influenza virus-resistant hemagglutinin protein monoclonal antibody labeled by a horse radish peroxidase, substrate reaction solution of an enzyme, positive and negative controls, cleaning solution and reaction stop solution. The kit has high specificity and sensitivity, is easy to operate, can be used for the clinical and laboratory detection of large-scale H1N1 swine influenza viruses and has wide application prospect.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to an anti-H1N1 swine influenza virus hemagglutinin protein monoclonal antibody, a hybridoma cell line and an antigen capture ELISA kit. Background technique [0002] Swine influenza (swine influenza, SI) is an acute, zoonotic respiratory infectious disease caused by influenza A (type A) virus in pigs or humans. It belongs to the family Orthomyxoviridae and the genus A(A) influenza virus. It is a single-stranded negative-sense RNA virus with a genome of about 13.6kb, consisting of 8 independent segments of varying sizes. Type A influenza virus contains 16 kinds of hemagglutinin proteins (H1-H16) and 9 kinds of neuraminidase proteins (N1-N9). Currently the most common subtypes are H1N1, H1N2 and H3N2, and many new virus variants and subtypes are still being discovered all over the world. [0003] The clinical symptom of swine flu is a sudden rise in body temperature (40-45°C)....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10C07K16/06G01N33/577G01N33/569G01N33/543C12R1/91
Inventor 朱忠武唐连飞肖家勇孟芳候义宏姜金林禹思宇欧阳振宇
Owner 湖南出入境检验检疫局检验检疫技术中心
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